GBS Bioinformatics Pipeline(s) Overview Getting from sequence files to genotypes. Pipeline Coding: Ed Buckler Jeff Glaubitz James Harriman Presentation: Terry Casstevens With supporting information from the coders.
Philosophy of the GBS Pipeline Why develop our own pipeline? Efficiency Only align unique fragments once. Data structures specific to these data types.
Three Pipelines Discovery Pipeline Requires a reference genome Multiple steps to get to genotypes Hands on tutorial is based on this pipeline Production Pipeline Uses information from Discovery Pipeline One step from sequence to genotypes UNEAK Pipeline For species without a reference genome Fei Lu will present this tomorrow at 9:30
Vocabulary Sequence File Text file containing DNA sequence and supplemental information from the Illumina Platform. Taxa An individual sample Key File Text file used to assign a GBS Bar Code to a Taxa GBS Tag DNA sequence consisting of a cut site remnant and additional sequence. GBS Bar Code A short known sequence of DNA used to assign a GBS Tag to its original Taxa
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
Raw Sequence (Qseq) HWI-ST397 0 3 68 15896 200039 0 1 GTCGATTCTGCTGACTTCATGGCTTCTGTTGACG HWI-ST397 0 3 68 15960 200043 0 1 GAGAATCAGCTTTTCCAACACCTTGAGTTTGAGT HWI-ST397 0 3 68 15831 200053 0 1 ATGTACTGCACCGTTGCAAGCGAGCACCACCAA HWI-ST397 0 3 68 15867 200049 0 1 CCAGCTCAGCCTGCATTCTTTCAAAAACTTCCAA HWI-ST397 0 3 68 15943 200048 0 1 GATTTTACTGCACATCGGTCTTGTCACACCAGCT HWI-ST397 0 3 68 15812 200062 0 1 TCACCCAGCATCACGCCCCTTCACATCCAGTAAA HWI-ST397 0 3 68 15888 200067 0 1 CTTGACTGCCACCATGAATATGTGTTCCAAGTGC HWI-ST397 0 3 68 15969 200067 0 1 CCACAACTGCTCCATCTTTTCCATGAGACATTGC HWI-ST397 0 3 68 15786 200078 0 1 GTATTCTGCACACGAATCAGCTGAGACACCAATT HWI-ST397 0 3 68 15830 200072 0 1 AATATGCCAGCAGTTAAGAGAGTTCAAGATCCAG HWI-ST397 0 3 68 15863 200073 0 1 CTCCCTGCGGGTGCGCGCGACCCATCTTCAGTT HWI-ST397 0 3 68 15762 200088 0 1 TGGTACGTCTGCGGAATGGCGTTTTTTATGCCTT HWI-ST397 0 3 68 15903 200085 0 1 GGACCTACTGCCCAAGAACGGCTCACCCATCAT HWI-ST397 0 3 68 15921 200082 0 1 GAGAATCAGCGTGTACGGGGCACGGGGTGACT HWI-ST397 0 3 68 15984 200085 0 1 TTCTCCAGCCGCATGGGCCGGAGACCAGAGAG HWI-ST397 0 3 68 15788 200096 0 1 GCGTCAGCAAATGCCCCAACAGCCAAGTCAGCA HWI-ST397 0 3 68 15842 200099 0 1 TAGGCCATCAGCTGACTTCCCGGGTGTGGAGAA HWI-ST397 0 3 68 15876 200105 0 1 GGACCTACTGCCGGCGGGACGAAAGCGGTTGT HWI-ST397 0 3 68 15937 200097 0 1 CTCCCTGTTGAAGCATGTGCAAAAGAGCTTGTTC HWI-ST397 0 3 68 15958 200102 0 1 CGCCTTATCTGCCCTCGCCGGTCATGGGGAGTG
Raw Sequence (Qseq) HWI-ST397 0 3 68 15896 200039 0 1 GTCGATTCTGCTGACTTCATGGCTTCTGTTGACG HWI-ST397 0 3 68 15960 200043 0 1 GAGAATCAGCTTTTCCAACACCTTGAGTTTGAGT HWI-ST397 0 3 68 15831 200053 0 1 ATGTACTGCACCGTTGCAAGCGAGCACCACCAA HWI-ST397 0 3 68 15867 200049 0 1 CCAGCTCAGCCTGCATTCTTTCAAAAACTTCCAA HWI-ST397 0 3 68 15943 200048 0 1 GATTTTACTGCACATCGGTCTTGTCACACCAGCT HWI-ST397 0 3 68 15812 200062 0 1 TCACCCAGCATCACGCCCCTTCACATCCAGTAAA HWI-ST397 0 3 68 15888 200067 0 1 CTTGACTGCCACCATGAATATGTGTTCCAAGTGC HWI-ST397 0 3 68 15969 200067 0 1 CCACAACTGCTCCATCTTTTCCATGAGACATTGC HWI-ST397 0 3 68 15786 200078 0 1 GTATTCTGCACACGAATCAGCTGAGACACCAATT HWI-ST397 0 3 68 15830 200072 0 1 AATATGCCAGCAGTTAAGAGAGTTCAAGATCCAG HWI-ST397 0 3 68 15863 200073 0 1 CTCCCTGCGGGTGCGCGCGACCCATCTTCAGTT HWI-ST397 0 3 68 15762 200088 0 1 TGGTACGTCTGCGGAATGGCGTTTTTTATGCCTT HWI-ST397 0 3 68 15903 200085 0 1 GGACCTACTGCCCAAGAACGGCTCACCCATCAT HWI-ST397 0 3 68 15921 200082 0 1 GAGAATCAGCGTGTACGGGGCACGGGGTGACT HWI-ST397 0 3 68 15984 200085 0 1 TTCTCCAGCCGCATGGGCCGGAGACCAGAGAG HWI-ST397 0 3 68 15788 200096 0 1 GCGTCAGCAAATGCCCCAACAGCCAAGTCAGCA HWI-ST397 0 3 68 15842 200099 0 1 TAGGCCATCAGCTGACTTCCCGGGTGTGGAGAA HWI-ST397 0 3 68 15876 200105 0 1 GGACCTACTGCCGGCGGGACGAAAGCGGTTGT HWI-ST397 0 3 68 15937 200097 0 1 CTCCCTGTTGAAGCATGTGCAAAAGAGCTTGTT HWI-ST397 0 3 68 15958 200102 0 1 CGCCTTATCTGCCCTCGCCGGTCATGGGGAGTG
Key File Flowcell Lane Barcode DNASample LibraryPlate Row Column LibraryPrepID PlateName 81PVTABXX 2 CTCC Sample_1 1 A 1 1 Plate_A 81PVTABXX 2 TGCA Sample_2 1 A 2 2 Plate_A 81PVTABXX 2 ACTA Sample_3 1 A 3 3 Plate_A 81PVTABXX 2 CAGA Sample_4 1 A 4 4 Plate_A 81PVTABXX 2 AACT Sample_5 1 A 5 5 Plate_A 81PVTABXX 2 GCGT Sample_6 1 A 6 6 Plate_A 81PVTABXX 2 TGCGA Sample_7 1 A 7 7 Plate_A 81PVTABXX 2 CGAT Sample_8 1 A 8 8 Plate_A 81PVTABXX 2 CGCTT Sample_9 1 A 9 9 Plate_A 81PVTABXX 2 TCACC Sample_10 1 A 10 10 Plate_A 81PVTABXX 2 CTAGC Sample_11 1 A 11 11 Plate_A 81PVTABXX 2 ACAAA Sample_12 1 A 12 12 Plate_A 81PVTABXX 2 TTCTC Sample_13 1 B 1 13 Plate_A 81PVTABXX 2 AGCCC Sample_14 1 B 2 14 Plate_A 81PVTABXX 2 GTATT Sample_15 1 B 3 15 Plate_A 81PVTABXX 2 CTGTA Sample_16 1 B 4 16 Plate_A 81PVTABXX 2 ACCGT Sample_17 1 B 5 17 Plate_A 81PVTABXX 2 GTAA Sample_18 1 B 6 18 Plate_A 81PVTABXX 2 GGTTGT Sample_19 1 B 7 19 Plate_A
Fragment from GBS library: GBS Tags Barcode adapter Cut site Insert Cut site Common adapter Good reads: (only the first 64 bases after the barcode are kept) typical read: Barcode Cut site Insert (first 64 bases) short fragment: Barcode Cut site Insert (<64bp) Cut site Common adapter chimera or partial digestion: Barcode Cut site Insert (<64bp) Cut site 2 nd Insert
Fragment from GBS library: GBS Tags Barcode adapter Cut site Insert Cut site Common adapter Good reads: (only the first 64 bases after the barcode are kept) typical read: Barcode Cut site Insert (first 64 bases) short fragment: Barcode Cut site Insert (<64bp) Cut site chimera or partial digestion: Barcode Cut site Insert (<64bp) Cut site
Fragment from GBS library: GBS Tags Barcode adapter Cut site Insert Cut site Common adapter Good reads: (only the first 64 bases after the barcode are kept) typical read: Barcode Cut site Insert (first 64 bases) short fragment: Barcode Cut site Insert (<64bp) Cut site chimera or partial digestion: Barcode Cut site Insert (<64bp) Cut site Rejected reads: Barcode Cut site Common adapter Not matching barcode and cut site remnant Contains N in first 64 bases after the barcode adapter dimer
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
Tag Counts With information from the key file, each sequence file is processed, tags are identified and counted. If a tag is shorter than 64 bases it is padded. The tags and counts are put into a tag count file for each sequence file. QseqToTagCountsPlugin / FastqToTagCountsPlugin
Master Tag Counts The individual tag count files are merged into a master tag count file. A minimum count is specified at the merge stage to exclude tags with low counts (likely sequencing errors). MergeMultipleTagCountsPlugin
Conversion of Tags to Fastq Sequence aligners do not work with the tag count file format. In preparation for the alignment step, the tag count file is converted to fastq format. TagCountsToFastqPlugin
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
Tag Alignment / TOPM The GBS pipeline uses an external aligner to do the initial alignment. The current version uses bowtie2 which produces the alignment in the SAM format. bowtie2 We convert the SAM file into our tags on physical map format (TOPM) SAMConverterPlugin
TOPM
So Far We Have Identified and counted GBS tags. Converted tag counts file to fastq. Aligned the tags to a reference. Converted the alignment to TOPM.
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
Tags by Taxa In this step we identify which tags are present in which taxa. Original Sequence Files Key File Master Tag Count File Recently migrated to HDF5 file format. Efficient storage Large data sets SeqToTBTHDF5Plugin
Tags By Taxa Additional Operations If many TBTs have been created they are merged into 1 TBT. Taxa that were sequenced multiple times are merged. The TBT table is pivoted in preparation for SNP calling. ModifyTBTHDF5Plugin
GBS Discovery Pipeline Discovery Sequence Tags by Taxa Tag Counts TOPM SNP Caller
SNP Calling Files used in SNP Calling TOPM TBT Some Key Settings mnf MinimumF (inbreeding coefficient) mnmaf Minimum Minor Allele Frequency mnmac Minimum Minor Allele Count mnlcov Minimum Locus Coverage TagsToSNPByAlignmentPlugin
HapMap rs# alleles chrom pos strand SgSBRIL067:633Y5AAXX:2:C9 SgSBRIL019:633Y5AAXX:2:C3 S1_2100 A/G 1 2100 + N N N N N N N R N A N S1_2163 T/C 1 2163 + N N N N N N T C T T N S1_13837 T/G 1 13837 + N N N N N N N G N N T S1_14606 C/T 1 14606 + N N C N N N T T T T C S1_2061 T/A 1 20601 + T N N N N N N A N N N S1_68332 C/T 1 68332 + N N N N N N N N N N N S1_68596 A/T 1 68596 + A N N N N N N N N A N S1_69309 G/A 1 69309 + N G N N N N N A N N N S1_79955 T/G 1 79955 + N T G T T N T T N N N S1_79961 T/G 1 79961 + N T T T T N T T N N N S1_80584 G 1 80584 + N N N N N N N N N N G S1_80647 C/T 1 80647 + N N N N N N N C N N C S1_81274 T/G 1 81274 + N N N N N N T G N N N S1_108834 G/A 1 108834 + N N N N N N N N N N N S1_112345 T/G 1 112345 + N N N N N N K T N N N S1_115359 C/T 1 115359 + N N N N N N T C N T S1_115362 T/C 1 115362 + N N N N N N N C N N N S1_115405 G/A 1 115405 + G G A N N G G G G N S1_115516 T/G 1 115516 + N N T N N N T T N N T S1_116694 A/G 1 116694 + N A G N N N G A N N N S1_119016 C/T 1 119016 + N N N N C N N C N N N S1_155366 T/C 1 155366 + N T N N N N
Discovery Fastq GBS Discovery pipeline Tags by Taxa Tag Counts TOPM SNP Caller
Discovery Fastq GBS Discovery pipeline Tags by Taxa Tag Counts TOPM SNP Caller Filtered
Production Pipeline
Why another pipeline? The last maize build (30000 taxa) with the discovery pipeline took over 3 months. Most common alleles have been identified after the first few discovery builds. Use the information from the discovery pipeline to call SNPs in new runs quickly. Improve efficiency and automate.
GBS Bioinformatics Pipelines Discovery Production Fastq Fastq Tags by Taxa Tag Counts TOPM SNP Caller
Discovery Fastq Production Fastq Tags by Taxa Tag Counts TOPM TagsOnPhysicalMap (TOPM) SNP Caller
GBS Bioinformatics Pipelines Discovery Production Fastq Fastq Tags by Taxa Tag Counts TOPM SNP Caller Filtered
GBS Bioinformatics Pipelines Discovery Production Fastq Fastq Tags by Taxa Tag Counts TOPM TOPM SNP Caller Filtered
GBS Bioinformatics Pipelines Discovery Production Fastq Fastq Tags by Taxa Tag Counts TOPM TOPM SNP Caller Filtered
GBS Bioinformatics Pipelines Discovery Production Fastq Fastq Tags by Taxa Tag Counts TOPM TOPM SNP Caller Filtered
Running the Production Pipeline Required Files: Sequence file (fastq or qseq) Key file Production TOPM TASSEL 3 Standalone & RawReadsToHapMapPlugin Running the Pipeline: One lane processed at a time HapMap files by chromosome ~20 minutes
Testing Production Pipeline Compared HapMap files produced by Discovery Pipeline and Production Pipeline Site Comparison: Discovery 48,139 Production 47,676 Difference due to maximum 8 alleles 99.98% correlation of genetic distance matrices
Next Steps In Pipeline Development Hierarchical Data Format supports very large data sets and complex data structures. Working to fuse TOPM, TBT, Keyfile, and Pedigree File into one HDF5 repository. Continued improvements to SNP caller. Ability to use tags not present in the reference.